Interaction between CENP-C and CENP-A takes place at centromere positions.

<p>HEK293 cells transfected with plasmids expressing the chimeric protein combinations indicated at the top of each column A–E (A: N-GFP::HA::CENPC and HA::CENPA::C-GFP; B: N-GFP::HA::CENPC; C: HA::CENPA::C-GFP; D: N-GFP::HA::CENPCΔ890-943 and HA::CENPA::C-GFP; E: N-GFP::HA::CENPCΔ890-943). After fixation cells were immunolabelled with a monoclonal anti-HA antibody and polyclonal antibodies used as centromere markers (A and D: anti-CENP-B; B and E anti-CENP-A; C: anti-CENP-C). Row I shows complementing GFP (green); row II shows merge between complementing GFP and the centromeric marker (red); row III shows HA-tagged chimeric proteins (cyan); row IV shows merge between HA-tagged chimeric proteins and the centromeric marker; row V shows merge between centromeric marker and DNA (blue). F: diagrams of the chimeric proteins expressed in cells. G: quantification of colocalization between the green signal due to complementing GFP and the centromeric marker in cells that coexpress HA::CENPA::C-GFP and N-GFP::HA::CENPC full length or the truncated mutant N-GFP::HA::CENPCΔ890-943. 10 to 15 nuclei for each experiment were analyzed and the data expressed as ratio between GFP signal corresponding to centromeres and number of visible centromeres. Statistical significance have been checked with T-student test and indicated in the graph. The bright green punctate signals in panel AI indicate efficient GFP complementation in cells coexpressing N-GFP::HA::CENPC and HA::CENPA::C-GFP. The green signals in AI well correspond to centromere positions as indicated by merge in panel AII and to HA-tagged proteins shown in panels AIII-IV. The green signal shown by cells expressing respectively only N-GFP::HA::CENPC or HA::CENPA::C-GFP is very low (BI) or absent (CI) although the chimeric HA-tagged proteins are expressed. In cells expressing N-GFP::A::CENPCΔ890-943 and HA::CENPA::C-GFP few green signals are visible corresponding to centromere position (green and red in DI–II) suggesting lower interaction when CENP-C Mif2 homology domain III is deleted although N-GFP::HA::CENPCΔ890-943 localizes at centromeres (EIII–IV).</p

Both Mif2p homology domains II and III target centromeres.

<p>(A) The indicated chimeric HA::CENP-C proteins were expressed in human HEK-293T cells and revealed by an anti-HA monoclonal antibody (red signal), while endogenous CENP-B was detected with an anti-CENP-B polyclonal antibody (green signal). Co-localization of the HA::CENP-C proteins and CENP-B is shown in yellow in the merged image. In the diagram, bars describe the different CENP-C truncated proteins as compared to the wt protein; Mif2p homology domain II (light grey), Mif2p homology domain III (dark grey), central DNA binding domain (dotted box), HA-tag (black box). Localization of proteins within the nuclei has been determined by epifluorescent microscopy. (B) Centromere localization of HA::CENP-C fusion proteins in interphase nuclei. For each mutant at lest 100 transfected cells were scored for colocalization of HA and CENP-B signals.</p

Sequence similarity among Mif2p homology domains of CENP-C orthologs.

<p>Amino acid sequences of human CENP-C Mif2p homology domains II and III, contained in the HA::638/819 and HA::760/943 constructs respectively, are compared to those of orthologous CENP-C proteins. Mif2p homology domains II and III are indicated in light grey and dark grey, respectively. Percent conservation is represented as follows: 100% conservation (red), 90% conservation (blue), 80% conservation (cyan); 70% conservation or less (white). Multiple sequence alignments of the CENP-C protein families were built with ClustalW, Multialin version 5.4.1 and T-Coffee and edited by hand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005832#pone.0005832-Corpet1" target="_blank">[64]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005832#pone.0005832-Notredame1" target="_blank">[66]</a>. Accession Numbers: Human CENP-C (GenBank M95724); Chimpanzee CENP-C (GenBank XM517266-7); Dog CENP-C (GenBank XM532388); Cow CENP-C (GenBank XM598358); Mouse CENP-C (GenBank U03113); Rat CENP-C (GenBank AAU04621.1); Chicken CENP-C (GenBank BAA24110.1); Sheep CENP-C (GenBank AAA79099.1); <i>A. thaliana</i> CENP-C (GenBank AAU04629.1); <i>S. cerevisiae</i> Mif2p (GenBank NP012834.1).</p

Pc binds to the <i>gcm</i> promoter region.

<p>(A–B) Association of the <i>gcm</i> and <i>gcm2</i> loci with PcG proteins. (A) Levels of Polycomb (Pc) binding and H3K27me3 at the <i>gcm</i> or <i>gcm2</i> gene locus and control regions (GlacAT and Rp49) in <i>Drosophila</i> embryos were determined by quantitative ChIP (qChIP) experiments. Results are represented as percentage of input chromatin precipitated. The standard deviation was calculated from two independent experiments. (B) Organization of the <i>gcm-gcm2</i> loci, extent of the used transgenic constructs (blue lines) and ChIP-on-chip binding profiles of indicated PcG proteins and histone marks in <i>Drosophila</i> embryos. Data were extracted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003159#pgen.1003159-Schuettengruber4" target="_blank">[33]</a>. The plots show the ratios (fold change) of specific IP versus mock IP assays. Significantly enriched fragments (P-value<1×10⁻⁴) are shown in red. Black bars indicate the location of primers used for qChIP analysis. (C,D) Eyes from flies carrying an empty mini-<i>w⁺</i> transgenic vector (C) or a mini-<i>w⁺</i> vector including a 9 kb <i>gcm</i> transgene (D). Flies heterozygous for the transgene are on the left, homozygous ones on the right. (E–H) Polytene chromosome immuno-FISH experiments performed on the <i>gcm</i> locus and PcG proteins. Immuno-FISH staining in wt (<i>w¹¹¹⁸</i>) flies (E,F) or flies carrying a transgene including a 9 kb region upstream of the <i>gcm</i> TSS (G,H), with anti-Pc (E,G) or anti-Ph (F,H) antibodies. Nuclear DAPI labeling in blue. Right panels show higher magnifications of the inserts. Double labeling (E,F) with a <i>gcm</i> probe (E″,F″) and anti-Pc antibody (E′″) or anti-Ph antibodies (F′″) detects colocalization (arrow) at one Pc or Ph binding site in wt; transgenic animals (G–H) show a second site of colocalization. (G″–G′″, H-Hb′″). Colocalization of <i>gcm</i> and Ph (arrow) in wt (D) and in the transgenic line (F).</p

The <i>Pc</i> mutation rescues the <i>gcm</i> LOF phenotype.

<p>(A,B) Schematic drawings showing the pupal wing at (A) 29 and (B) 16 hr APF (in all panels, anterior the top, distal to the right. Inset in (A) indicates the region shown in (C–F,I,N). L3-v, L3-1 and L3-3 indicate the sensory neurons. (C–R) Immunolabeling of 24 hr APF wings: <i>gcm-Gal4:UAS-GFP/+</i> (<i>gcm-Gal4/+</i>), considered as wt (C–H), <i>gcm-Gal4</i> (I–M) and <i>gcm-Gal4;Pc/+</i> (N–R). Anti-GFP labeling (green) reflects <i>gcm</i> expression, anti-Repo (red) marks glia and anti-Elav (blue) marks neurons. (C–H) Bracket in (E) indicates the glial cells produced by the L3-v sensory organ precursor; bracket in (F) indicates the three proximal neurons (L3-v, ACV, E1). White arrowhead indicates the L3-v neuron. Insets indicate the regions shown at higher magnification (C,I,N). (G,H) The L3-v GP produces several GFP+/Repo+ cells (arrows). In mutant wings (I–M), the L3-v lineage produces only one GFP+ cell (J,M), which does not express Repo (K), but Elav (L,M asterisk indicates the ectopic neuron). In double <i>gcm</i> and <i>Pc</i> LOF wings (N–R), several GFP+ cells (O,R) express Repo (P) and no ectopic neurons were observed (Q). (S) Quantitative data on the fate transformation phenotype at different stages. (T–V) Immunolabeling in 9 hr APF wings: <i>gcm-Gal4/+</i> (T–T″); <i>gcm-Gal4</i> (U–U″) and <i>gcm-Gal4;Pc/+</i> (V–V″). In all genotypes, one GFP+ cell produced by the L3-v lineage is visible (T,U,V). In the heterozygous wing, this cell expresses Repo (T′) and not Elav (T″). In <i>gcm-Gal4</i> (U), the GFP+ cell does not express Repo (U′), but expresses Elav (U″). In the double <i>gcm</i> and <i>Pc</i> LOF wing, the GFP+ cell (V, empty arrowhead) expresses Repo (V′) and Elav (V′). Scale bars: C–F,I,N = 100 µm; G,H,J–M,O–R,U–W″ = 10 µm.</p

Genetic screen for <i>gcm^Pyx</i> modifiers and interactions with TrxG and PcG proteins.

<p>(A) Drawing of an adult notum. Small and large dots represent microchaetae and macrochaetae, respectively. Macrochaete symbols to the right. (B–E) Adult nota from wt (WT; B), <i>gcm^Pyx/+</i> (C), <i>gcm^Pyx</i>/suppressor deficiency (D), <i>gcm^Pyx</i>/enhancer deficiency (E) flies. Dfs = Deficiencies. Scale bar = 200 µm. Histograms present the average number of bristles per heminotum (y-axis) in different genotypes (x-axis). In all figures, average values are indicated +/− SEM (bars); <i>P-</i>values from t-test are indicated in the following way: *** (<i>P</i>≤10⁻³), ** (<i>P</i>≤10⁻²), * (P≤5×10⁻²). <i>Pyx</i> stands for <i>gcm^Pyx</i>. (F) Deficiencies deleting <i>brm</i>, (<i>Df(3L)brm11</i> and <i>Df(3L)th102</i>), as well as the <i>brm</i> mutation. <i>P-</i>values vs. <i>gcm^Pyx</i>/+: <i>gcm^Pyx</i>/+; <i>Df(3L)brm11/+</i> (8,9×10⁻⁶); <i>gcm^Pyx/+</i>; <i>Df(3L)th102/+</i> (0,02); <i>gcm^Pyx</i>/+; <i>brm/+</i> (4,2×10⁻⁷). (G) <i>gcm^Pyx</i> interaction with <i>trxG</i> genes. <i>P-</i>values vs. <i>gcm^Pyx</i>/+: <i>gcm^Pyx</i>/+; <i>brm/+</i> (4,2×10⁻⁷); <i>gcm^Pyx</i>/+; <i>osa/+</i> (0,002); <i>gcm^Pyx</i>/<i>UAS-osa; hsGal4/+</i> (0,0007); <i>gcm^Pyx</i>/+; <i>brm</i>/<i>osa</i> (3,4×10⁻⁸); <i>gcm^Pyx</i>/+; <i>trx/+</i> (0,009); <i>gcm^Pyx/+</i>; <i>ash1</i>/+ (0,01); <i>nej</i>/+; <i>gcm^Pyx/+</i> (6,9×10⁻⁷); <i>gcm^Pyx</i>/+; <i>E(bx)/+</i> (1,3×10⁻⁵). (H) <i>gcm^Pyx</i> interaction with <i>PcG</i> genes. Color code indicates members of the same complex (dark gray: PRC1, pale gray: PRC2, black: PRC recruiter). <i>P-</i>values vs. <i>gcm^Pyx</i>/+: <i>gcm^Pyx</i>/+; <i>Pc¹</i>/+ (6,2×10⁻⁶); <i>gcm^Pyx</i>/+; <i>Pc³</i>/+ (0,0022); <i>gcm^Pyx</i>/+; <i>Pc¹⁵</i>/+ (1,5×10⁻¹¹); <i>gcm^Pyx</i>/+; <i>E(z)</i>/+ (0,001); <i>gcm^Pyx</i>/<i>esc</i> (2,6×10⁻⁸); <i>gcm^Pyx</i>/<i>psq</i> (0,03). (I) Summary of the tested TrxG and PcG mutations. From left to right: the biochemical complex, the genes within the complex, the mutant phenotype over <i>gcm^Pyx</i> (No – no effect; S – suppressor; E – enhancer) and the large deficiency phenotype (nt – gene region not covered by the kit).</p

Small deficiencies tested in the secondary screen.

*<p>only upstream region.</p><p>From the primary screen to the genes. From left to right, columns indicate the name of the large deficiencies identified in the primary quantitative screen, the name of the small deficiencies in that region, their cytology, the phenotype observed over <i>gcm^Pyx</i> (No – no effect; S – suppressor; E – enhancer), the name and phenotype of putative interactors genes analyzed over <i>gcm^Pyx</i>.</p

PcG genes control glia proliferation.

<p>(A–E) Quantitative analysis of glia at L3 vein position. Graphs comparing animals of different genotypes for the number of glia present on the L3 vein by 20 hr APF. The Y-axis indicates the percentage of wings showing a given number of glia; the X-axis, the number of glia expressing the Repo protein. Color-code is used to distinguish the compared genotypes: (A) wt vs. <i>gcm-Gal4/+</i>, (B) wt vs. <i>gcm-Gal4</i>, (C) <i>gcm-Gal/+</i> vs. <i>gcm-Gal4</i>, (D) <i>gcm-Gal4</i> vs. <i>gcm-Gal4</i>; <i>Pc/+</i>. (A) In wild type wings, the number oscillates between 5 and 7, whereas in <i>gcm-Gal4/+</i> wings it oscillates between 3 and 9 cells. (B,C) <i>gcm-Gal4</i> homozygous animals carry fewer Repo labeled cells and less variation (from 3 to 6) than heterozygous animals (from 3 to 9). This is also reflected by the presence of one peak value for homozygous animals and two for heterozygous animals. (D) Note that <i>gcm-Gal4</i>; <i>Pc/+</i> animals show an increase of glial cell number compared to that observed in <i>gcm-Gal4</i> animals. (E) The graph shows the distribution around the average of the number of Repo+ cells in the different genotypes as indicated by the color code. (F–H) Quantitative analysis of glia at L1 vein position. Graphs comparing animals of different genotypes for the number of glia present on the L1 vein by 24 hr APF. The Y-axis indicates the percentage of wings showing a given number of glia; the X-axis, the quantitative range of Repo expressing cells. Color-code is used to distinguish the compared genotypes: (F) wt vs. <i>Pc/+</i> or vs. <i>E(z)/+</i>, (G) <i>Pc/+</i> vs. <i>E(z)/+</i> or vs. <i>Pc/E(z)</i>. (F) Most wild type animals show from 50 to 60 glia. (G) Note that most <i>Pc/E(z)</i> double heterozygous animals show higher number of glia (from 70 to 80 Repo+ cells) when compared to single heterozygous animals. This is confirmed by more than 20% of wings showing over one hundred Repo+ cells on L1 vein. (H) The graph shows the distribution around the average of the number of Repo+ glia at the L1 vein position in the different genotypes as indicated by the color-code. (I) Quantitative analysis of pupal wings showing a double Repo/PH3+ cell indicating glia proliferation.</p

<i>gcm</i> is overexpressed in <i>Pc</i> mutants.

<p>(A–J) <i>In situ</i> hybridization with a <i>gcm</i>-specific probe. (A,B,D,E) 19 hr APF wings: <i>gcm</i> is expressed at the L1 nerve position (L1) and in the so-called twin sensilla of the margin (TSM) in wt (A) as well as in <i>Pc/+</i> animals (D); by 24 hr APF, <i>gcm</i> is no more expressed in wt (B), but persists in <i>Pc/+</i> wings (E) (asterisk indicates a non-specific signal). (C,F) <i>gcm</i> expression in the embryonic brain (arrowhead) and in the ventral cord (brackets) fades by stage 14 in wt (C), but persists in <i>Pc</i> mutants (F) (lateral views, anterior to the left). (G–J) optic lobe partial projection (anterior to the top; scale bar = 100 µm): in wt (G), <i>gcm</i> is expressed at the position of the lamina glial cell precursor (GPC) area (arrows); <i>gcm</i> expression in <i>Pc/+</i> (H), in <i>brm/+</i> (I) and in <i>brm</i>, <i>trx/+</i> double mutants (J). Note that we focused on early third instar larvae, when the first burst of expression takes place in the GPC region. At that time, <i>gcm</i> is just starting being expressed in the other territories that have been previously described as <i>gcm</i> positive <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003159#pgen.1003159-Soustelle2" target="_blank">[39]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003159#pgen.1003159-Soustelle3" target="_blank">[40]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003159#pgen.1003159-Chotard1" target="_blank">[43]</a>. (K) Schematic representation of optic globe <i>gcm</i>-dependent lamina glial lineages. In blue, the GPCs. In green, differentiating and migrating glial cells (direction shown by the arrows). (L) Schematic representation of the areas of <i>gcm</i> expression (red) in the GPC region, based on the above <i>in situ</i> analyses.</p